Reactivation of resolved hepatitis B virus (HBV) infection is increasingly recognized in patients with severe immunosuppression. We monitored seven patients with pretransplant antibodies to hepatitis B surface antigen (anti-HBs) and hepatitis B core antigen (anti-HBc) for HBV reactivation after allogeneic haematopoietic stem cell transplantation (allo-HSCT). Reverse seroconversion (from anti-HBs to HBsAg) was observed in six recipients occurring 12, 14, 16, 22, 31 and 39 months after allo-HSCT, respectively. The only patient without HBV reactivation had the highest pretransplant anti-HBs titre and died after the shortest follow-up period (25 months). A novel HBV surface mutant (D144G/G145E) was isolated from one recipient of stem cells from a donor vaccinated against HBV. Another surface mutant (P142L/G145R) was detected in a recipient from a non-immune donor. Serum ALT elevation was measured in only two of the six patients with viral reactivation, followed by spontaneous clearance of HBsAg in one of them. Antiviral treatment reduced viral load in five patients, but the emergence of YMDD motif polymerase mutations resulted in lamivudine resistance in two patients. In conclusion, the risk of reactivation of a resolved HBV infection is close to 100% in allogeneic stem cell recipients and vaccination of the donor does not always warrant reliable protection.